Summary

Immunohistochemistry is a branch of histochemistry. It is a technique of in situ detection of the distribution of antigens (or antibodies) in tissues by labeled specific antibodies (or antigens). All antigenic substances in tissue cells, such as peptides, hormones, neurotransmitters, cytokines, receptors and surface antigens, can be displayed by immunohistochemistry, so they are widely used in basic and clinical research.

Classification of immunohistochemical staining methods

1.According to the types of labeled materials, such as fluorescent dyes, radioisotopes, enzymes (mainly horseradish peroxidase and alkaline phosphatase), ferritin, colloidal gold, etc., it can be divided into immunofluorescence method, radioimmunoassay, enzyme labeling method and immune gold silver method.

2.According to the dyeing steps, it can be divided into direct method (also known as one-step method) and indirect method (two-step, three-step or multi-step method); compared with direct method, the sensitivity of indirect method is much higher.

3.According to the binding mode, it can be divided into antigen antibody binding, such as peroxidase anti peroxidase (Pap) method and affinity binding method, such as ovalbumin biotin peroxidase complex (ABC) method, Streptomyces avidin peroxidase (SP) method.

Objective

The macromolecules, such as peptides, hormones, neurotransmitters, cytokines, receptors, surface antigens and so on, were localized, and their functions were further studied.

Principle

1.Immunofluorescence method

Immunofluorescence is the first established immunohistochemical technique. Based on the principle of specific binding of antigen and antibody, the known antibody is labeled with fluorescein, which is used as a probe to examine the corresponding antigen in cells or tissues and observe under the fluorescence microscope. When the fluorescein in the antigen antibody complex is irradiated by the stimulated luminescence, it will emit a certain wavelength of fluorescence, which can determine the location of a certain antigen in the tissue, and then quantitative analysis can be carried out. Because of its high specificity, sensitivity, rapidity and simplicity, immunofluorescence technology is widely used in clinical pathological diagnosis and examination.

2.Immunoenzyme labeling method

Immunoenzyme labeling is a technique developed in 1960s after immunofluorescence. The basic principle is that the antibody labeled by enzyme interacts with tissue or cell, and then the substrate of enzyme is added to form colored insoluble products or particles with certain electronic density. The antigenic components on cell surface and in cell are studied by light microscope or electron microscope. Immunoenzyme labeling is the most commonly used technique at present.

3.Immune colloidal gold technique

Immunocolloidal gold technology is based on colloidal gold as a special metal particle as a marker.Colloidal gold is the hydrosol of gold, which can adsorb protein rapidly and stably, but has no obvious effect on the biological activity of protein. Therefore, using colloidal gold labeled primary antibody, secondary antibody or other molecules (such as Staphylococcus a protein) that can specifically bind to immunoglobulin as probes, we can qualitatively, localize and even quantitatively study the antigens in tissues or cells. Because colloidal gold has different size particles and high electron density, immune colloidal gold technology is particularly suitable for single label or multi label localization of immunoelectron microscopy. Because the colloidal gold itself is light to dark red, it is also suitable for light microscopic observation. For example, it is easier to observe under light microscope.

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